There are millions of patients worldwide afflicted with cancer who are treated with radiation therapy. Radiation therapy is used as a primary therapy or in combination with surgery and/or chemotherapy and/or hormone therapy. Most common cancer types may be treated with radiation therapy in some way. The precise treatment or radiation dose depends upon the tumor type, location, stage, as well as the general health of the patient. In most cases, radiation therapy is given to patients for two to six weeks at a total dose of 50 Gy to 70Gy. High doses of radiation is required to kill all the cancer cells, but the radiation injures both the tumor cells as well as normal tissues that are in its path and treatment causes many side effects. Side effects include soreness, diarrhea, nausea, edema, infertility, fatigue, fibrosis, hair loss, dryness of mouth, or damage to salivary glands. In some cases, radiation itself can lead to the formation of cancer. Repetitive use high doses of radiation is limited because the chances of damage to vital organs such as the spinal cord increases as exposure to radiation increases. Thus, there is a need for therapeutic agents which can effectively reduce the dose of radiation required to treat cancer cells so that side effects may be minimized or eliminated and radiation therapy may be repeated as necessary.
Sphingolipids such as ceramide and sphingosine (SPH) are a class of apoptosis regulators in cancer cells. Ceramide inhibits proliferation and promotes apoptosis (10), while sphingosine-1-phosphate (S1P) is a key tumor-promoting lipid, responsible for tumor cell proliferation, migration and invasion (1). S1P is synthesized by phosphorylation of SPH. However, the formation of SW antagonizes the formation of ceramide. The opposing directions between the formation of ceramide and S1P is referred to as the “sphingolipid rheostat” and plays a pivotal role in regulating tumor growth (10) (FIG. 1). The levels of ceramide and S1P are regulated by sphingosine kinase-1 (SPK-1), whose overexpression has been shown to inhibit apoptosis (10). Studies have also shown that elevated levels of S1P and increased SPK-1 activity in cancers is due to the overexpression of SPH (2,3), while the reduction of SPK1 levels in cancer cells results in apoptosis of the cancer cells (10, 15).
SPK-1 activity has been shown to be upregulated in many types of cancers including various squamous cell carcinomas (SCC) such as head and neck, lung, bladder, ovary, prostate, and skin cancers. Likewise, cell lines from various types of cancers including the human breast cancer (MCF-7) (6-8); intestinal tumors (9); prostate adenocarcinomas (10); colon cancers (11); lung cancers (12); erytholeukemias (13); and bladder tumors (14) also exhibit SPK-1 overexpression.
French et. al. used various cancer cells and cell lines including human breast cells and breast, colon, lung, ovary, stomach, uterus, kidney and rectum tumors from patients, to demonstrate that SPK mRNA is over-expressed in cancer as compared to normal tissue in the surrounding area of the same organ (15). French used a chemical library of 16,000 compounds to screen for compounds that inhibited SPK overexpression. Four compounds were discovered, but not all of these compounds were specific for SPK inhibition, as some compounds inhibited other human kinases.
Nava, et al., showed a relationship between SPK-1 and resistance to irradiation in prostate cancer cell lines. SPK1 activity in radioresistant LNCaP cells were not affected by gamma-irradiation, but SPK1 levels of radiosensitive TSU cells were noticeably inhibited by gamma-irradiation. Radioresistant LNCaP cells were however, sensitized to gamma-irradiation when treated with TNF-alpha, which is known to decrease SPK1 levels (23).
Other pharmacological agents that have been shown to reduce the levels of SPK in tumor cells include phenoxodiol (16); dimethylsphingosine (DMS) (18); docetaxel and camptothecin (10); agents derived from marine bacterium B-5354 (19) and fungus (20); and sphingoside analogs (17). However, these pharmacological agents exhibit moderate levels of antitumor activity or exhibit toxicity that makes the use of pharmacological inhibitors undesirable. Thus, there is a need for inhibitors that are effective at inhibiting SPK overexpression, without producing undesirable side effects.
Small interfering RNAs (siRNAs) have been shown to specifically “knock out” or “silence” the gene of specific proteins and enzymes and are effective at inhibiting the overexpression of SPK without the toxicities and other undesirable side effects associated with the use of pharmacological inhibitors.
siRNAs have been shown to reduce SPK-1 activity and induce apoptosis in MCF-7 cells (21) and decrease cell viability in PC-3 cells (22).